With the recent completion of the human genome sequence, the sequencing of other vertebrate genomes has taken center stage. The sequencing of widely studied model organisms (e.g., mouse, rat, and zebrafish) is providing a valuable resource for future experimentation and important insights into vertebrate biology. Less clear is the relative value of other candidate genomes being considered for systematic sequencing, especially with regard to their potential contribution to the annotation and interpretation of the human genome sequence. To investigate such issues, we generated large blocks of orthologous sequence from multiple vertebrates for detailed comparative analyses;one such example is the ENCODE project which aims to identify all functional elements in the human genome. Specifically, the same targeted genomic regions from multiple vertebrate species are being isolated in large-insert bacterial artificial chromosome (BAC) clones and then sequenced. Efficient methods for designing orthologous hybridization probes and isolating BAC clones from the different species have been developed and implemented. Following characterization by several mapping methods, tiling paths of BACs are then selected and systematically sequenced. The establishment of this comparative sequence resource is facilitating the development of new computational tools for multi-species sequence comparisons, providing insight about the appropriate degrees of sequencing finishing that should be pursued in the sequencing of other vertebrate species, and revealing the benefits of sequencing species from a range of different evolutionary distances from human. Additional efforts by collaborating investigators involve determining complex genome architectures, which can only be accomplished through sequence data generated by BAC-based sequencing. NISCs BAC-based sequencing pipeline will focus on providing data for such projects. In addition to its inter-species sequence comparisons, NISC developed a second major sequence-production pipeline designed for performing intra-species (specifically, inter-human) sequence comparisons for medical research projects. In establishing and utilizing a PCR-based sequencing pipeline, NISC once again capitalized on its unique niche of being embedded within the broader NIH Intramural Program with its outstanding clinical research infrastructure. Many medical sequencing projects were completed that directly interface with well-established clinical researchers at other NIH Institutes and utilizes the NIH Clinical Center to study the molecular basis for common human diseases, with an emphasis on the detection and study of rare disease-associated variants. In the last year, NISC has implemented the most advanced Next Generation sequencing technologies to provide at least a thousand-fold greater sequencing capacity. This is providing a powerful, state-of-the-art resource to clinical researchers for the most advanced and complex studies possible. Together, the two pipelines of the NISC Comparative Sequencing Program should continue to produce data at the cutting edge of genomics research, exploring how large-scale DNA sequencing can be used to characterize the human genome and to understand the genetic basis for human health and disease. In addition, NISC is scaling up microbial survey and genome sequencing as part of the NIH Intramural Skin Microbiome Consortium. Sanger-based sequencing of 16S amplified PCR amplicons is transitioning to shorter, but vastly more numerous 16S sequences obtained through Roche 454 DNA sequencing. To complement the clinical sample survey sequencing, isolated reference genomes will be sequenced and finished to standards comparable with those established by the NIH Human Microbiome Project. Plans are underway to survey fungal inhabitants and whole genome sequencing of selected isolates as well. Finally, NISC is heavily engaged in implementing powerful new sequencing technologies into its pipelines. Extensive testing of available platforms has been ongoing, providing important insights about which new technologies are best-suited for full-scale implementation at NISC. These technologies are rapidly evolving and NISC will continue to monitor developments and implement those it deems most appropriate for the sequence data we produce for collaborating investigators. Virtually all sequencing projects will be affected by the implementation of these new technologies.